The standard method of genomic analysis for mutations and polymorphisms, including for CF, is the “dot-blot” method. Samples including target strands are spotted onto a nitrocellulose support, and then contacted with labeled probes complementary to the mutations or polymorphic regions. The labels allow detection of probe hybridization to immobilized complementary target sequences, as unbound labeled probes are removed by washing. In another method—a “reverse dot-blot format”—an array of oligonucleotide probes is bound to a solid support, and then contacted with a sample including target sequences of interest. See, e.g., U.S. Pat. No. 5,837,832.
Both methods of assaying mutations or polymorphisms have significant disadvantages. The dot-blot method is itself labor-intensive. It can also yield erroneous results due to the inaccurate reading of assay signals, usually done by autoradiography, which adds further labor, as the probes must be frequently re-labeled. The method described in U.S. Pat. No. 5,837,832 involves a complex and costly on chip synthesis of an array of oligonucleotides, an approach which is better-suited for large-scale genomic analysis and is neither practical nor cost-effective for diagnostic applications requiring only a limited but changing number of probes.
An assay method suitable for multiplexed analysis which avoids many of the problems associated with the above methods involves use of random encoded arrays of microparticles, where the encoding indicates the identity of an oligonucleotide probe molecule bound thereto. See U.S. patent application Ser. No. 10/204,799: “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays.” The bead array is contacted with labeled amplicons, generated from a patient sample, and the labels are then detected (if the labels are fluorescent, the detection can be with optical means) and the bound amplicons are identified by decoding of the array.
In a multiplexed hybridization assay, cross-hybridization among mismatched, but closely homologous, probes and amplicons can generate false positive signals. Thus, the assay should be designed to minimize such effects. A number of mutations and polymorphisms are significant only if they are homozygous, and therefore, to be useful in such cases, the assay must be capable of discriminating heterozygotes from homozygotes. Also, in determining the assay results, where both the encoding method for the beads and the determination of assay results is with optically detectable means, the encoding on the beads can cause spectral leakage, which can be affect the assay signal discrimination. A method of correcting for such spectral leakage is also needed.
Cystic fibrosis (“CF”) is one of the most common recessive disorders in Caucasians, with an occurrence of 1 in 2000 live births in the United States. Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are associated with the disease. The number of CFTR mutations is growing continuously and rapidly, and more than 1,000 mutations have been detected to date. See Kulczycki L. L., et al. (2003), Am J Med Genet 116:262-67. Population studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the CFTR amino acid sequence (designated ΔF508), is associated with approximately 70% of the cases of cystic fibrosis. This mutation results in the failure of an epithelial cell chloride channel to respond to cAMP (Frizzell R. A. et al. (1986) Science 233:558-560; Welsh, M. J. (1986) Science 232:1648-1650; Li, M. et al. (1988) Nature 331:358-360; Quinton, P. M. (1989) Clin. Chem. 35:726-730). In airway cells, this leads to an imbalance in ion and fluid transport. It is widely believed that this causes abnormal mucus secretion observed in CF patients, and ultimately results in pulmonary infection and epithelial cell damage. A number of mutations are associated with CF, and researchers continue to reveal new mutations associated with the disease. The American College of Medical Genetics (“ACMG”) has recommended a panel of 25 of the most common CF-associated mutations in the general population, especially those in Ashkenazi Jewish and African-American populations. A multiplexed hybridization assay for CF-associated mutations in the general population would test for this panel.